Note: Descriptions are shown in the official language in which they were submitted.
~ 1~';~9~9
This invention relates ~o novel N-(mercaptoacyl)-cysteines
(I) and to a process for their manufacture.
The novel N-~mercaptoacyl)-cysteines are compounds of formula I
Z - CO - NH - CH - COOH
SH CH2-SH
wherein Z is a group of formula CH3-CH , ~CH3)2C C . (CH2)2 - or CH2 = -
The compound of formula I can be prepared by debenzylating compounds of
formula IV
I - CO - NH - fH - COOH IV
S - CH2-c6H5 CH2-S-CH2-C6 H5
The compounds of formula I are also useful in the form of their
salts with pharmaceutically acceptable cations.
Compound ~I) and the intermediate (IV) have not been disclosed
in any literature. The compound (I) has, as to be described later, a strong
action of decreasing the mucus viscosity and therefore it is useful as an
agent for the liquefaction of sputum. The sulfhydryl compound ~I) is con-
sidered to liquefy sputum through the splitting of the mucoprotein disulfide
linkages.
Compounds-of present invention may be prepared by the following
process,
Benzylmercaptoacyl halide represented by ~he formula (II) reacts
with S-benzyl cysteine ~III) by the conventional method such as Schotten-
Baumann reaction to form N-~benzylmercaptoacyl)-S-benzylcysteine represented
by the formula ~IV). The resulting compound ~IV) reacts with reducing agent
to allow debenzylation to form N-~mercaptoacyl)-cysteine (I). Any reducing
agent of conventional types may be used, but metallic sodium in liquid am-
monia is preferable.
Z-CO-X NH2-CH-COOH
f
S-CH2-C6H5 CH2-S-CH2-C6H5
~II) (III)
- 2 - ~ ;
.. .
~ ' ' . .
^` 1079Z99
wherein Z is selected from the group consisting of CH3-CH~, (CH3)2C~,
~CH2)2'~and CH2~ and X is halogen.
Z-CO-NH-CH-COOH
S-CH2-C6H5 CH2-S-CH2 C6H5 (IV)
Z-CO-NH-CH-C00H
SH CH2-SH (I)
The compound (II) used in this reaction can be obtained by reacting ben`zyl-
mercapto carboxylic acid with halogenizing agent such as thionyl chloride in
usual method. Some of the compounds represented by the formula (II) and the
formula (III) have asymmetric carbon atom in their structures and the compound
(IV) or (I) obtained therefrom naturally has two asymmetric carbon atoms in
each compound. Accordingly, if compounds (II) and/or (III) are optically
active substances, compound ~IV) or (I) of optically active form is obtained.
If either of the two compounds ~II) and (III) is optically active substance,
diastereoisomer is formed. Inventors of the present invention have obtained
two kinds of compound by reacting racemlc form of the compound (II) wherein
Z is CH3-CH~ , with L-form of (III) and these two kinds of compound obtained
are considered to be in relation of diastereoisomer by judging from their
optical rotations.
The present invention is further illustrated by the following
examples and by the pharmacological study in vitro and toxicological study
in animals, but they are not to be construed as limiting the present invention.
Example 1
(1) Preparation of N-(2-benzylmercaptoisobutyryl)-S-benzyl-L-cysteine
(IV, Z=(CH3)2C~)
73.9g of S-benzyl-L-cysteine (III) was dissolved in 700 ml of 1 N
sodium hydroxide solution. The solution was cooled in an ice bath and stirred.
2-Benzylmercaptoisobutyryl chloride (II, Z=(CH3)2CJ), which was obtained by
reacting 63.lg of 2-benzylmercaptoisobutyric acid with 39.3g of thionyl
chloride was added dropwise to this solution. The resulting mixture was
- 1~7~Z~9
further stirred for one hour, acidified with hydrochloric acid and extracted
with ethyl acetate. The extract was washed with water, dried over sodium
sulfate and evaporated to dryness. The residue was chromatographed on sili-
cagel with benzeneethylacetate (1 : 1) as eluent. The eluate was evaporated
to dryness and oily residue weighed 46.9g, representing a yield of 74%, was
obtained.
(2) Preparation of N-~2-mercaptoisobutyryl)-L-cysteine ~I,
Z=~cH3)2c')
46.9g of the compound CIV, Z=~CH3)2C~) obtained in ~1) above was
dissolved in 500ml of liquid ammonia and 21.1g of metallic sodium was added
bit by bit with stirring. After completion of reaction, 59.4g of ammonium
chloride was added and thereafter the ammonia was removed by distillation.
Water was added to the residue to dissolve the solid. Water layer was separat-
ed, washed with ethyl acetate~ and acidified with hydrochloric acid under
i cooling. The precipitateSthus obtained were extracted with ethyl acetate.
- The extract was washed with water, dried over sodium sulfate and evaporated to
dryness. The product weighed 43.6g, representing a yield of 88%. After recry-
stallization from ethyl acetate, the desired compound melting at 139-140C
was obtained.
- [~]25 +32.3 ~c=l.0, ethanol).
Anal- Calcd- for C7H13N03S2 C, 37-65; H~ 5-87; N~ 6-27-
Found: C, 37.78; H, 5.86; N, 6.16.
j Example 2
~1) Preparation of N-(2-benzylmercaptopropionyl)-S-benzyl-L-cysteine
A and -B (IV, Z=CH3-CH~)
211g of S-benzyl-L-cysteine (III) was dissolved in 22 liter of 0.1
N sodium hydroxide solution, and to the solution 236g of 2-benzylmercapto-
propionyl chloride (II, Z=CH3-CH~) was added dropwise with cooling and stir-
ring. The resulting mixture was further stirred for one hour, acidified
with hydrochloric acid and extracted with ethyl acetate. The extract was
.: . . .
10'79Z99
washed with water, dried over sodium sulfate, and concentrated to form cry-
stals. The crystals were collected by filtration and thereafter washed with
small amount of ethyl acetate. It weighed 154g, representing a yield of 40%.
After recrystallization from ethyl acetate, N-(2-benzylmercaptopropionyl)-S-
benzyl-L-cysteine-A (IV, Z=CH3-CH~) melting at 111.5-112.5C was obtained.
~]D5: -135.9 (c=1.4, ethanol),
Anal- Calcd- for C20H23N3S2 C~ 61-69; ~ ; J
Found: C, 61.73; H, 5.92; N, 3.62.
The filtrate~ from which N-~2-benzylmercaptopropionyl)-S-benzyl-L-
cysteine-A was removed, was concentrated and the residue was chromatographed
on SiO2 with ethyl acetate. The eluate was evaporated to dryness, and oily
residue weighed 144g, representing a yield of 37%.
~]25: +46.2 ~c=5.2, ethanol). Oily N-~2-benzylmercaptopropionyl)-S-benzyl-
L-cysteine-B(IV, Z=CH3-CH~) was thus obtained.
(2) (i) Preparation of N-(2-mercaptopropionyl)-L-cysteine-A (I,
Z=CH3-CH~)
77.8g of N-(2-benzylmercaptopropionyl)-S-benzyl-L-cysteine-A was
dissolved in 2.5 liter of liquid ammonia. In small portions, 16.1g of metal-
lic sodium was added with stirring. After completion of reaction, 25g of
ammonium chloride was added and thereafter the ammonia was removed by distil-
lation. Water was added to the residue to dissolve the solid. Water layer
was separated, washed with ethyl acetate, and acidified with hydrochloric
acid with cooling. The precipitates thus obtained was extracted with ethyl
acetate. The extract was washed with water, dried over sodium sulfate, and
evaporated to dryness. The product weighed 39.3g, representing a yield of
94%. After recrystallization from ether, the desired compound melting at
114-115C was obtained.
r~] 2. +3.5~c=2.0, ethanol~
Anal. Calcd. for C6HllNO3S2: C, 34.45; H, 5.30; N, 6-70-
Found: C, 34.95; H, 5.36; N, 6.61.
- 5 -
.
9Z99
(2) (ii~ Preparation of N-(2-mercaptopropionyll-L-cysteine-B (I,
- Z-CH3-CH~)
Proceeding with 144g of N-~2-benzylmercaptopropionyl)-S-benzyl-L-
cysteine-B, 2 liter of liquid ammonia, 28g of metallic sodium and 37g of
ammonium chloride, according to ~2)-(i~ above, N-(Z-mercaptopropionyl)-L-
cysteine-B (I, Z=CH3-CH~ was obtained in a yield of 71g. (92 percent of the
theoretical amount~. After recrystallization from ethyl acetate, the desired
compound melting at 124-125C was obtained.
[~]D4: + 16.8 (c=1.7, ethanol).
10 Anal. Calcd. for C6HllNO3S2: C, 34.45; H, 5.30; N, 6-70-
Found: C, 34.67; H, 5.28; N, 6.77.
Example 3
By the methods described in the foregoing examples and using the
appropriate starting materials, the following compounds of formula (I) were
prepared: N-(3-mercaptopropionyl)-L-cysteine (I, Z=(CH2)2~). m.p. 97C.
~al25: +4.9 (c=l.0, ethanol).
6 11NO3S2: C, 34.45; H, 5.30; N, 6.70.
Fou~d: C, 34.45; H, 5.37; N, 6.70.
N-mercaptoacetyl-L-cysteine (I, Z=CH2~). m.p. 113C. (dec.).
20 ~ ~25: +24 9 (c=l.0, ethanol).
Anal. Calcd. for C5HgNO3S2 C, 30.77; N, 4.65; N, 7 18.
Found: C, 30.80; H, 4.71; N, 7.24.
Mucolytic activity is evaluated by determining the reduction in vis- -
; cosity of egg white or mucin in vitro. L-Cysteine ethyl ester hydrochloride,
.. , ~
L-cysteine methyl ester hydrochloride and acetyl-L-cysteine are used as test
compounds for comparison.
` (1) In vitro determination of the reduction in viscosity of egg white
Egg white, which is pooled in sufficient quantity to supply material
for all the determinations, is gently blended in a mixer for 5 minutes. 9ml
~ 30 portions of this material are put into a 50-ml beaker and the test solution
.'
.
,,. . . . ... . . :
.
1~79Z~9
in a volume of lml containing various concentrations of test compound (test
compound is dissolved in distilled water and the solution IS adjusted to pH -
8.0 with 1 N sodium hydroxide, while, distilled water is used as a control)
is added. The mixture is then shaked for 20 minutes at 37C. Viscosity of
the resulting solution is determined by Ostwald's viscosimeter. Results
shown in Table 1 are per cent reduction in relative viscosity at various cCn-
centrations of the test compound used.
(2) In vitro determination of the reduction in viscosity of mucin
According to the method presented by Sheffner et al. (Ann, N.Y.
Acad. Sci., Vol. 106, P. 298-310, 1963), bacteriological mucin (Nakarai
Chemical Co., Ltd.) is dissolved in Sorensen's phosphate buffer ~pH = 8.0)
and 1% mucin solution is prepared. O.9ml portions of the solution are put
into test tubes and test compounds ~prepared in the same manner (1), previous-
ly described, except that the buffer solution is used for control) are added
to them in a concentration lOmM. The mixture is then shaked for 1 hour at
37C. Viscosity of the resulting solution is determined by Ostwald's
viscosimeter. Results shown in Table 1 are per cent reduction in relative
viscosity at lOmM of the test compounds used.
(3) Changes in the mucolytic activity of the test compounds treated
with rabbit serum.
O.lml of test compound (prepared in the same manner as (1) previous- -
ly described) and O.9ml of rabbit serum are put into a 50-ml beaker and the
mixture is shaked for 30 minutes at 37C. 9ml of egg white is then added to
it and it is shaked for another 20 minutes. Yiscosity of the resulting
solution is determined by Ostwald's viscosimeter. Results shown in Table 2
are per cent reduction in the relative viscosity of egg white at lmM of the
test compounds after treatment with or without rabbit serum.
-
-- 7 --
- .. , . . :
' ' , , : : . ~ .
1~)79Z'99
Table 1
Te6t compound Final concent- Per cent reduction
ration ~M) in relati~e viscosity
egg white ~ucin
~-(2-mercapto- 0.1 23.1 ~ 1.4
cysteine 1.0 38.3 ~ 2.2
10.0 5~.0 + 1.022.5 ~ 1.5
~-(2-~ercapto- 0.1 24.9 ~ 1.5
cy6te~ne-A 1.0 41.2 ~ 2.1
10.0 56.4 ~ 1.626.6 ~ 1.5
~-(2-~ercapto- 0.1 24.9 ~ 1.4
rotpi~O yl)_~_ 1.0 3~ 2.6
10.0 57.7 + 1.020.1 + 0.4
n-(3-~ercapto- 0.1 22.8 + 0.1
propionyl)-L- 1. a 36.7 ~ 1.6
10.0 54.5 ~ 4.728.1 ~ 0.5
~-mercapta- 0.1 26.1 ~ 4.1
cysteine 1.0 39.8 t 3.0
10.0 60.0 ~ 4.431.8 t 1.5
L-cy~teine 0.1 25.1 ~ 3.5
ethyl e6t~r
hydrochloride1.0 42.0 ~ 1.1-
10.0 59.9 ~ 1.219.7 + 1.5
L-cy~teine 0.1 31.1 + 5.6
. methyl ester 1 0 45.5 + 2.0
hydrochloride --
10.0 60.6 + 2.417.8 + 0.2
~-acetyl-L- 0.1 11.0 ~ 2.6
cy6teine 1.0 29.5 + 2.5
10.0 ~ 49.0 + 1.84.8 ~ 2.5
Results are'the mean values + S.E. of 3 to 5 experiments.
- 10 -
' ' ' ' ' : , . ~ ~ ' ' '. ' ' .............................. ' '
, ~ . . .- , . . . . .
1079Z99
,
Table 2
Test compoun~ Final Per ce~t reductio~ in Reduction
con_ relative ~isco~ity of ~n the
cent- egg ~hite mucolytic
ratlon untreated treated acti ity
with rabbit with rabbit
6erum seruo
~-(2-mercapto-
isobutyryl)-~- 1.0 38.3 + 2.2 32.g + 1.014.1
cy~teine
n-(2-mercapto-
propionyl)-L- 1.0 41.2 + 2.1 30.0 ~ 1.027.2
cysteine-A
~-(2-mercapto-
prop~onyl)-~- 1.0 38.1 + 2.6 31.1 ~ 3.818.8
cystei~e-~
~-(3-mercapto-
propio~yl)-L- 1.0 36.7 ~ 1.6 38.7 ~ 2.6-5.4
cy~teine
~-mercapto-
acetyl-L- 1.0 39.8 ~ 3.0 35.9 ~ 0.39.8
cysteine
~-cysteine
~th~l ester 1.0 42.0 + 1.115.4 ~ 1.863.3
hydrochloride
L-cysteine
~ethyl e~ter 1.0 45.5 ~ 2.07.0 ~ 1.284.6
hydrochloride
~-acetyl-L-
cy~teine 1.0 29.5 ~ 2.5 18.5 + 0.837.3
., .
~ Result6 are the ~ean values i 5.E. of 3 to 5 experiments.
. ,
~ .
_ _ _
~ 9
. ~
1079Z99
Toxicological study
Among the compounds (I) o present inventio~, ~-~2-
~erca~toisQbutyryl)-L_cy6teine and ~-(2-mercaptopropio~yl)-
L-cy6teine-A are emamined i~ re6pect to t~eir acute
Toxicity. The result~ are shown in Table 3.
Table 3
Test co~pound Ani~al Route of ad- Acute Toxicity
u~ed ~ini~tratio~ (LD50)
~-(2-mercapto- i.p. 22g5sg/kg
~sobutyrylJ-L- ~ou6e
cy~teine i.v. 989.6~g/kg
~-t2-~ercapto-
propionyl)-~- mou~e i.p~ 2092mg/kg
cy~teine-A
Animal u~ed
~-week-old male mice of ddy strain ~body weight
:24-28g~ are u6ed. They are fed ior one week befare
.
experiment and animals exhibiting abnormal growth during
th~6 period are excluded.
,
Te~t 601utian
Tect compound i6 added to phy~iological 6aline and
the re6ulting solution i~ neutralized with 60diu~ hydr-
oxide tc for~ 5~ 601ution.(pH 7.0).
O~ser~at~o~
: Aiter ad~ini6tra~ion of te~t ~olution~ general
~ymptom6 and death6 which are caused dur~ng ~even day~
are ob~erved The LD50 value6 are calculated according
. to the method of Litchfield and Wilcoxon.
' 10
.
.
. . .
~0'79Z9~
As evide~t from the pharmacological study and the
toxicological study the compound (I) of thi inYention
i6 useful a~ an agent ror the liquefaction of 8putum.
In the ca6e of medical treatment of an adult, the do~e
per day ra~ge~ 100 - 600mg depending upon the methad and
the irequency of adminl6tration. The compound maX be
~ admini6tered orally or directly to trachea, or inhaled
- in aero601 form.
A compo6itio~ for ~edicine of the compound of thi~
in~ention may be processed, for oral ad~i~istratio~,
i~to tablet~, powder or encapulated for~ so that they
are ea~ily ab~orbed fro~ stomach or intestine6, and they
ay be proce6sed i~to ~uch for~ in view of convenience.
For thi6 purpo~e, binding age~t such as ~elati~, orbitol,
polyvinyl pyrrolidone; ~or~ing agent such a6 laGtose,
6tarche, calcium phosphate; lubricatlng agent ~uch as
mag~e~iu~ ctearate, talc; collapsing agent ~uch a~
carboxy methyl celluloe calcium may be added. The co~-
po~ition may be proces~ed into an~aqueous solut1on of
the compound aloue or its salt 6uch as 60d~um salt, for
the ue of aero~ol or for injectio~ use.
Foll~wings are the exa~ple6 of for~ulations i~ which
(2-~ercaptoiobutyryl~-L-cysteine i6 used a a ~odel
but aid compound may be changed with otherco~pounds of
formula (I) ln accordance with forms proce~sed.
~, .
'~
1~79Z99
(1) For oral administration
~a) Tablet for~
: ~-(2-mercaptoi60butyryl)-L-cy6tei~e lOOmg
Ethyl cellulo~e 5~g
Cry6talline cellulo6e 80~6
~ Carboxy~ethyl cellulose 7mg
- ~agne6ium ~tearate ~g
Total 240mg
Tablet may be coated with fil~-coating which i~
u6ually adopted. It may be ~urther coated with
6ugar-coatin~.
(b) Granular for~
N-(2-mercaptoisobut~ryl)-~-cy6teine lOOmg
PolyYinyl pyrrolidone 25mg
Lacto6e 365mg
Talc- lOm~
~: Total 500
(c) Pow~er forR
h-(2-~ercaptoisobutyryl)-L-cy~teine laO~g
~acto~ 500mg
Starch 370~g
Colloidal 6ilica 30
Total 1000
12
- . . . . ..
. .
1079Z99
; (d) Encap~ulated for~
~-(2-mercaptoisobutyryl)-L-cystelne lOOmg
Lacto~e . 32mg
; Crystalline cellulo~e 56mg
Colloidal silica 2mg
Total l90mg
(2~ Aero~ol ~orm and in~ection form
250m~ o~ N-(2-~ercaptoisobutyryl)-L-cyfitei~e are
contained in 5m~ o~ aqueous solution of p~ 6.5 - 7.0, a~
the ~odiu~ ~a}t for~ of the compound.
.
'
- 13
. - ~
